Guanine nucleotide regulation of hormone-sensitive adenylate cyclase is mediated through a component (G-protein) distinct from both the hormone receptor and catalytic cyclase. Cholera toxin, in the presence of (32P)NAD, specifically radiolabels a 42,000 Mr subunit of the G-protein. Partial proteolytic digestion of the radiolabeled cholera toxin substrate from pigeon erythrocyte membranes produces similar peptide maps with the native membrane bound form and after solubilization with Lubrol PX. These results indicate that the peptide, which has been shown to be an elongated intrinsic membrane protein that binds little or no detergent after solubilization, is largely on the intracellular side of the membrane supporting the hypothesis that it is a "stalked" intrinsic membrane protein. In the absence of added guanine nucleotide, treatment of labeled membranes with proteases results in the production of five detectable trypsin specific fragments and one chymotrypsin specific fragment derived from the 42,000 Mr radiolabeled peptide. The addition of GTP gamma S or GDP beta S to the membrane incubation results in distinguishable differences in both tryptic and chymotryptic peptide maps of the 42,000 Mr protein. Hormone-receptor interactions affect the formation of specific fragments suggesting a direct effect of receptor on the G-protein. The ability to detect specific conformational changes of the G-protein by these techniques is being used to study specific hormone-responsive states of adenylate cyclase.